title: 08D
description: Protocols: 100 plants of the Brassica napus winter-type accession BnASSYST-120 (Darmor) were grown in a field in Merelbeke, Belgium (50째58'24.9\"N 3째46'49.1\"E) in a 10-by-10 grid pattern with 0.5 m spacing within and between rows. At 81 days after sowing (2016-11-28), rosette leaf 8 of 62 individual plants was cut off where the petiole first lacked conspicuous laminar tissue (looking from the leaf tip toward the base), and washed with DEPC-treated and sterilized water. The midvein was removed with scissors and the residual leaf material was folded into a 50 ml tube and stored in liquid nitrogen on the field. All leaf samples were harvested in a ~1-hour time span (13:25-14:27). All samples were sourced from individual plants grown in the same field under uncontrolled field conditions. ds cDNA was prepared using the Maxima H Minus Double-Stranded cDNA Synthesis Kit (#K2561, Thermo Fisher Scientific) to a concentration of ~17-38 ng/ul in 10mM Tris-Cl buffer (pH 8.5) at a minimum volume of 30ul. (~0.6 - 1.1 ug total). Total RNA was extracted using the guanidinium thiocyanate-phenol-chloroform extraction method using TRI-reagent (Thermo Fisher Scientific) followed by DNA digestion using the RQ1 RNase-free DNase kit (Promega). Library preparation (500bp average insert size) was performed at the University of Missouri Genomics Technology Core using the Illumina TruSeq DNA PCR-Free Library Prep Kit according to the protocol described in (Bivens and Zhou, 2016)